1. Field of the Invention
The present invention relates to a multilayer analysis sheet for analyzing liquid samples, and more particularly, it is concerned with a multilayer analysis sheet which enables quantitative determination of a specific chemical component in a liquid sample using a dry-type process which does not require the precise measurement of a definite volume of liquid sample and the weighing of and subsequent preparation of essential reagent(s). As used herein, the terminology "liquid sample" refers to a sample containing water as a solvent.
2. Description of the Prior Art
Multilayer analysis sheets are known which can be used to determine some specific chemical components contained in a liquid sample with ease and that, with high speed in a dry-type process. For instance, the specific examples of such analysis sheets are described in Japanese Patent Application (OPI) Nos. 53888/74 (U.S. Pat. Nos. 3,992,158), 137192/75 (U.S. Pat. No. 3,983,005), 40191/76 (U.S. Pat. No. 4,042,335), 3488/77 (U.S. Pat. No. 4,006,403), 131786/77 (U.S. Pat. No. 4,050,898), 131089/78 (U.S. Pat. No. 4,144,306), 29700/79 (U.S. Pat. No. 4,166,093) and 34298/79 (British Patent Application GB No. 2,000,869A) (the term "OPI" as used herein refers to a "published unexamined Japanese patent application"), U.S. Pat. Nos. 4,110,079 and 4,132,528, Clinical Chemistry, Vol. 24, pp. 1335-1350 (1978), and so on. Such multilayer analysis sheets have a common format, wherein a spreading layer capable of spreading liquid samples, layers containing reagents essential to the analysis, and so on are laminated in advance on a support, and, upon the actual chemical analyses using these sheets, quantitative analysis can be conducted through only two basic procedures. One procedure involves allowing a drop of the sample liquid to be examined to adhere onto the sheet, and the other is to evaluate the extent of change in color using a densitometer. Therefore, these procedures are referred to as dry chemical analyses, as they do not require procedures which are indispensable for conventional methods, such as: arrangement of test tubes; preparation, volume-measurement, and addition of reagent solutions; accurate weighing-out of samples; and so on.
The basic structure of the multilayer chemical analysis sheet of the type described above is constructed of a support, a reagent layer and a sample spreading layer, which are arranged in this order. The reagent layer is obtained by spreading reagent(s) incorporated in a binder like gelatin in a form of thin layer, which may have a monolayer structure or a multilayer one. The multilayer structure is constructed of a first reagent layer, the second reagent layer and so on, wherein reagents are contained separately, being classified in the order of reaction, and optionally, it can involve a detecting layer, a dye-receiving layer, and the like. In addition, interlayers such as a radiation-blocking layer, a barrier layer and the like can be provided between the spreading layer and the reagent layer, or between each pair of layers containing different reagents respectively. The sample spreading layer is arranged at the outermost position of an analysis sheet, and corresponds to the face to which liquid samples are to be adhered. The function of this layer is to supply a liquid sample to the reagent layer at an approximately constant volume per unit area regardless of its applied volume, that is to say, this layer acts as a spreader for a liquid sample. The action of spreader layer, then, is simply to allow a liquid sample placed on the sample spreading layer in a measured volume of x .mu.l, 2x .mu.l, 3x .mu.l . . . to spread on the sample spreading layer in proportion to the volume of the sample put thereon through the spreading action inherent in the layer; namely, to spread so as to cover an area of y cm.sup.2, 2y cm.sup.2, 3y cm.sup.2 . . . , respectively and consequently, to render the quantity of the sample to be supplied to the reagent layer per unit area approximately constant. This means that a liquid sample can be analyzed quantitatively without precise measurement of the volume thereof upon analysis, and this has an important significance.
The mechanism of spreading is not fully understood, but it is theorized that spreading results from and is limited by a combination of forces such as hydrostatic pressure of a liquid sample, capillary action within the spreading layer, surface tension of the sample, wicking action of layers in fluid contact with the spreading layer, and the like. As will be appreciated, the extent of spreading is dependent in part on the volume of liquid to be spread. However, it should be emphasized that the uniform concentration obtained with spreading is substantially independent of liquid sample volume and will occur with varying degrees of spreading. As a result, elements of this invention do not require precise sample application techniques. However, a particular liquid sample volume may be desirable for reasons of preferred spread times or the like. Because the elements of this invention are able to produce quantitative results using very small sample volumes that can be entirely taken up within a conveniently sized region of the spreading layer (e.g., one square centimeter), there is no need to remove excess moisture from the element after application of a liquid sample. Further, because spreading occurs in the spreading layer and the spread substance is provided to the fluid contacting reagent layer and without apparent substantial lateral hydrostatic pressure, there is not the "ringing" problem often seen with prior analytical elements when soluble reagents were used.
The spreading layer need only produce a uniform concentration of spread substance per unit area at its surface facing a layer with which the spreading layer is in contact.
Liquid sample spreading layers having such a sample spreading action as described above are described in detail in the aforementioned patent specifications and literature, which state that non-fibrous porous media alone are effective for the use as the layer possessing the above-described spreading action.
Examples of such non-fibrous porous medium include brush polymers (that is, membrane filters), diatomaceous earth, dispersions obtained by dispersing porous substances like microcrystalline materials (e.g., microcrystalline cellulose (Avicel, trademark of FMC Corporation)) in binders, porous aggregates formed by allowing fine spherical beads of glass or resin to adhere to one another in point-to-point contact, and so on. It is necessary for these non-fibrous porous media to have an isotropic porous form, that is to say, a form in which voids are distributed uniformly in all directions of the medium, as stated in U.S. Pat. No. 3,992,158.
There are two methods employable for the formation of the spreading layer consisting of such non-fibrous isotropically porous medium having the described liquid sample-spreading action. The first method involves lamination of an isotropically porous sheet, such as a commercially available membrane filter, on the reagent layer or a radiation-blocking layer, and subsequent adhesion of the sheet thereto. The nature of the radiation-blocking layer is well described in U.S. Pat. No. 4,042,335. This method is disadvantageous from technical and economic points of view, since the membrane filter is fragile and expensive. The second method involves coating a material capable of forming an isotropic porous medium layer; for example, material containing as a main component a solution of cellulose acetate in an acetone-dichloroethane (1:1) solution mixture; the same material containing additionally diatomaceous earth; a material prepared by dispersing glass beads of 80 to 120 mesh in a small amount of gelatin, and so on. The material is coated on the reagent layer, and upon subsequent drying of the layer coated under appropriate conditions the layer coated can be transformed into a homogeneous porous layer. No specific means is required for the drying. Usually, the drying is carried out by blowing air or an inert gas such as nitrogen gas, or by spontaneous drying, at temperatures of about 15.degree. to 80.degree. C., preferably 20.degree. to 50.degree. C. Many technical difficulties arise in the actual preparation according to the second method. For example, in manufacturing the porous spreading layer, it is technically difficult to control the voids contained in each product so as to obtain a uniform material and that, settled size, arrangement, volume ratio and so on. Moreover, in some cases it happens that reagents contained in the reagent layers are extracted with solvents used in the coating materials to cause diffusion of the reagents into the spreading layers. Further, when liquid samples containing proteins in high concentration, such as sera, are to be examined, what is more important is the defect that the sample-spreading action turns out to be essentially non-quantitative in the sample spreading layers made up of known non-fibrous porous media, because the spreading varys appreciably depending upon the protein content in the liquid sample on the layer. Furthermore, when whole blood samples are being tested, the sample spreading layers of the types described above suffer from the defect that the sample-spreading action therein turns out to be even less quantitative because it varys to a great extent depending upon the content of a solid component in addition to that of proteins.